-
Isolation, Structure, Synthesis and Cytotoxicity of an
UnprecedentedFlupirtine Dimer
René Csuka, Sven Sommerwerka, Jana Wiesea, Christoph Wagnerb,
Bianka Siewerta,Ralph Klugea, and Dieter Ströhla
a Martin-Luther-Universität Halle-Wittenberg, Bereich
Organische Chemie, Kurt-Mothes-Str. 2,D-06120 Halle (Saale),
Germany
b Martin-Luther-Universität Halle-Wittenberg, Bereich
Anorganische Chemie,Kurt-Mothes-Str. 2, D-06120 Halle (Saale),
Germany
Reprint requests to Prof. Dr. René Csuk. Fax: 0049 345
5527030.E-mail: [email protected]
Z. Naturforsch. 2012, 67b, 1297 – 1304 / DOI:
10.5560/ZNB.2012-0258Received October 2, 2012
A previously unknown dimer of the well-established analgesic
flupirtine has been found, and itsstructure was revealed by ESI-MS,
NMR spectroscopy and an independent synthesis. Thus, startingfrom
2-amino-6-chloro-3-nitro-pyridine the target compound was obtained
in a four-step synthesis.Key-step of this synthesis is a
nickel-mediated aryl-aryl coupling. The dimer 4 did not show any
cy-totoxicity, and its IC50 values were > 30 µm for all six
human cancer cell lines and mouse fibroblastsused in this
study.
Key words: Flupirtine, Dimerization, Aryl-Aryl Coupling
Introduction
Neurodegenerative diseases are the sixth-leadingcause of death
in Europe and North America. World-wide up to 40 million people
suffer from these diseases.Recently, neuroprotective properties
have been cred-ited to flupirtine (1), ethyl
N-[2-amino-6-(4-fluoro-phenylmethyl-amino)pyridin-3-yl]carbamate
(Fig. 1).Flupirtine has been used since 1984 as a centrallyacting
non-opiate analgesic [1]. In addition, it reducesmuscle-tone, and
it does not show the side effects ofnonsteroidal anti-inflammatory
drugs or of opiates.
Recently, flupirtine came twice in the focus ofrenewed
scientific interest. First, it was discussedfor treating memory
impairment and sensorimotoricshortfalls usually associated with
Gulf War Veteran’sIllness [2]. This disease affected ca. 25% of
thealmost 700.000 veterans of the Persian Gulf Warof 1990/1991.
Second, its neuroprotective propertiesmake 1 an interesting
candidate for the treatmentof neurodegenerative diseases, e. g.
Alzheimer’s andParkinson’s as well as Creutzfeld-Jacob disease [3,
4].In addition, fibromyalgia [5] has been treated success-fully
applying flupirtine. During our own studies on
neuroprotection we became interested in 1 and deriva-tives
thereof.
Results and Discussion
Flupirtine has been in medicinal use in Germanyfor more than
twenty years. Recently, the quantifi-cation of 1 and its related
compounds (e. g. impuri-ties resulting either from synthesis or by
decomposi-tion during storage) in pharmaceutical dosage formsby
UPLC has been reported [6]. Several years ago,Bednarski et al. [7]
re-examined some aspects of themetabolism of 1 and revealed the
formation of flupir-tine dimers 2 and 3 (Fig. 1); the structure of
the dimerswas deduced by interpretation of HRMS data. Forthese
dimers quasi-molecular ions m/z = 607.258937and m/z = 607.259010
matching [M+H]+ with an em-pirical formula M = C30H33F2N8O4 were
detected [7].
To shorten our synthesis [8, 9] of flupirtine analogswe decided
to use commercial 1 as a suitable start-ing material. Thus,
commercial samples of 1 (as itsmaleate) were bought. Their
analytical investigationshowed the presence of a dimeric compound
withm/z = 607.2 (ESI-MS, cation-sensitive mode) corre-
© 2012 Verlag der Zeitschrift für Naturforschung, Tübingen ·
http://znaturforsch.com
mailto:[email protected]
-
1298 R. Csuk et al. · An Unprecedented Flupirtine Dimer
Fig. 1. Structure of flupir-tine (1), of proposed in vitrodimers
2 and 3, as well as ofthe novel dimer 4.
sponding to [M+H]+ with M = C30H32F2N8O4; in ad-dition a
quasi-molecular ion m/z = 304.1 ([M+2H]+)was detected. However, no
dimers with an empir-ical formula C30H33F2N8O4 [7] were detected
inthese samples. Isolation of this novel dimer by semi-preparative
HPLC yielded enough material for its in-vestigation by NMR. From
the 1H, 19F and 13C NMRspectra the dimer was assigned structure 4
(Fig. 1).Thus, compound 4 is characterized in its 19F NMRspectrum
by the presence of only one signal at δ =−120.1 ppm. This suggests
a symmetrical dimer. Thesignals for an ethyl carbamate group are
found in the13C NMR spectrum at δ = 14.6 and 60.1 ppm. The 13CNMR
spectrum also shows the presence of four qua-ternary carbons in the
pyridine ring. Since there is stilla primary amino group present
(as indicated by the IRspectrum), the dimer has to possess a
central C–C bondconnecting the two pyridine rings. From the number
ofquaternary carbons in the pyridine ring and their chem-ical
shifts in the NMR spectrum, the connecting bondbetween the two
monomers has to be at position C-5 ofthe pyridine. To verify this
proposed structure, we setout for an independent synthesis (Scheme
1).
Thus, reaction of 2-amino-6-chloro-3-nitro-pyridine(5) with
4-fluorobenzylamine (6) [10, 11] for 10 h un-der reflux gave the
known compound 7 [8, 12] in ex-
cellent yield. Compound 7 is characterized in its 19FNMR
spectrum by the presence of a signal at δ =−115.9 ppm showing JF,H
= 8.9 and 5.5 Hz, respec-tively. Bromination of 7 with NBS in the
presenceof ammonium acetate [13] provided the bromo com-pound 8 in
almost quantitative yield. Compound 8was reduced by Zn/NH4Cl [14]
to yield the diamine9. The reduction of 8 using Zn/NH4Cl proceeds
withbetter results than the well-established reduction us-ing
Raney-Nickel/H2 at elevated temperatures and highpressures [8, 9].
Under these harsh conditions impure 9is obtained that has to be
re-crystallized several times.As an alternative, the use of Pd/H2
was suggested [10].The diamine 9 was transformed in situ [15, 16]
intothe corresponding N3-ethylcarbamate 10 by reactionwith ethyl
chloroformate/triethylamine. As previouslyshown by Paradisi et al.
[17, 18], the reaction of 2,3-diaminopyridines with ethyl
chloroformate proceedsregioselectively at the meta position when
the condi-tions are mild, and the temperature is kept low. No
di-acylation was observed.
From the reaction, however, a by-product 11 wasisolated in 10%
yield. The colorless solid showed inits 1H NMR spectrum the
presence of two ethyl car-bamate moieties (e. g. δ = 1.40 and 1.45
ppm for themethyl groups); the matching carbonyl groups were
-
R. Csuk et al. · An Unprecedented Flupirtine Dimer 1299
Scheme 1. Synthesis of compound 4. a) 2-Propanol, NEt3, reflux,
10 h, 98.8%; b) NBS, NH4OAc, CH3CN/THF, 0 ◦C, 3 h,98.6%; c) NH4Cl,
Zn, EtOAc, H2O, 1 h, T < 35 ◦C, quant.; d) NEt3, ClCO2Et, 0 ◦C,
2 h, 10 (80.5%), 11 (10%); e) NEt3,NiCl2, PPh3, Zn, THF, 60 ◦C, 2
d, 69%.
detected in the 13C NMR spectrum at δ = 149.9 and148.2 ppm. The
13C NMR spectrum revealed also thepresence of an extra carbonyl
group at δ = 146.0 ppm.There are four quaternary carbons in the
pyridine ring,and C-2 of the pyridine ring shows a shifting of |∆δ
|=14 ppm to higher field. From these data the struc-ture of 11 was
deduced. To corroborate this struc-ture, suitable crystals were
grown and subjected toa single-crystal X-ray analysis whose results
affirmedthe structure of 11 (Figs. 2 and 3) unambiguously [19].As
depicted in Fig. 3, two molecules of 11 are con-nected via a
symmetrical bifurcated hydrogen bridge,
thus resulting in a infinitive chain along the crystallo-graphic
glide plane [1̄01]. This interaction can be con-sidered as medium
strong; no further intermolecularinteraction is present.
Interestingly enough in the for-mation of 11 no
6-bromo-5-[(4-fluorobenzyl)amino]-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one
[18] wasformed other than the dicarboxylate 11.
Aryl-aryl bond formation can be accomplished bymany methods
[20], Stille and Suzuki couplings be-ing used quite often and very
successfully. Nickel-catalyzed homo-coupling reactions have been
stud-ied since the early 1970s and shown to be very
-
1300 R. Csuk et al. · An Unprecedented Flupirtine Dimer
Fig. 2. Molecular structure of 11 in the crystal with atom
la-belling scheme (50% probability ellipsoids; H atoms
witharbitrary radii).
Fig. 3. The bifurcated intermolecular hydrogen bond in
thecrystal structure of 11.
efficient for the synthesis of biaryls. Even thoughzero-valent
nickel reagents are generally
sensitive,tris(triphenylphosphine)nickel(0) can be generated insitu
using the Tiecco/Testaferri [21] modification ofKende’s procedure
[22]. The dimerization reaction of10 was crucial and failed in DMF
[23, 24] or pyri-dine [25] as a solvent and gave only low yields
of
the dimer. In these solvents a fast de-bromination re-action
took place; the main product was flupirtine (1).Coupling of 10 with
NiCl2/PPh3 in the presence of tri-ethylamine in THF, however,
advanced nicely and gavedimeric 4 in 68.8% isolated yield as an
off-white solid.Compound 4 showed in its ESI-MS spectrum a
cationwith m/z = 602.2 corresponding to a quasi-molecularion [M+H]+
and a set of signals in its 1H, 19F and13C NMR spectra
corresponding well with its dimericstructure. Compound 4 obtained
by synthesis provedto be identical in every aspect to the material
isolatedfrom the commercial drug. Since 4 has been foundas an
impurity in commercial samples of the drug in-tended for human use
(although in a rather low concen-tration of < 0.1%) we became
interested in its cyto-toxicity. Testing of 4 in a colorimetric
sulforhodamineassay [26] using six human cancer cell lines and
mousefibroblasts (NiH 3T3) gave IC50 > 30µM for each
cellline.
Experimental Section
Cell lines and culture conditions
The human cancer cell lines 8505C, A2780, A549, MCF-7, 518A2,
HT29 and mouse fibroblasts NiH 3T3 were in-cluded in this study.
Cultures were maintained as monolay-ers in RPMI 1640 (PAA
Laboratories, Pasching/Germany)supplemented with 10%
heat-inactivated fetal bovine serum(Sigma/Germany) and
penicillin/streptomycin (PAA Labora-tories) at 37 ◦C in a
humidified atmosphere of 5% CO2/95%air.
Cytotoxicity assay [26]
The cytotoxicity of the compounds was evaluated usingthe
sulforhodamine-B (SRB) (Sigma Aldrich) microculturecolorimetric
assay. In short, exponentially growing cells wereseeded into
96-well plates on day 0 at the appropriate celldensities to prevent
confluence of the cells during the periodof experiment. After 24 h,
the cells were treated with serialdilutions of the compounds (0 –
100 µM) for 96 h. The fi-nal concentration of DMSO or DMF solvent
never exceeded0.5%. The percentages of surviving cells relative to
untreatedcontrols were determined 96 h after the beginning of drug
ex-posure. After a 96 h treatment, the supernatant medium fromthe
96 well plates was discarded, and the cells were fixedwith 10% TCA.
For a thorough fixation, the plates were al-lowed to rest at 4 ◦C.
After fixation, the cells were washed ina strip washer. The washing
was done five times with waterusing alternate dispensing and
aspiration procedures. After-wards the plates were dyed with 100 µL
of 0.4% SRB (sul-forhodamine B) for about 20 min. The plates were
washed
-
R. Csuk et al. · An Unprecedented Flupirtine Dimer 1301
with 1% acetic acid to remove the excess of the dye and al-lowed
to air dry overnight. 100 µL of 10 mM Tris base so-lution were
added to each well, and absorbance was mea-sured at 570 nm (using a
96 well plate reader, Tecan Spectra,Crailsheim/Germany). The IC50
values were calculated ap-plying the two-parametric Hill slope
equation.
Synthesis and analysis
Reagents were bought from commercial suppliers andused without
any further purification. Melting points weremeasured with a Leica
hot stage microscope and were notcorrected. NMR spectra were
recorded on Varian Gemini200, Gemini 2000 or Unity 500
spectrometers at 27 ◦C withtetramethylsilane as an internal
standard, δ values are givenin ppm and J in Hz. Mass spectra were
taken on a Finni-gan MAT TSQ 7000 (electrospray, voltage 4.5 kV,
sheathgas nitrogen) instrument. Elemental analyses were measuredon
a Foss-Heraeus Vario EL unit. IR spectra were recordedon a
Perkin-Elmer FT-IR spectrometer Spectrum 1000 andUV/Vis spectra on
a Perkin-Elmer unit, Lambda 14. TLCwas performed on silica gel
(Merck 5554, detection by UVabsorption). Solvents were dried
according to usual proce-dures.
2-Amino-6-(4-fluorobenzylamino)-3-nitropyridine (7)
To a suspension of 2-amino-6-chloro-3-nitro-pyridine (5,47.00 g,
0.27 mol) in 2-propanol (240 mL), triethylamine(39.5 g, 0.39 mol)
and 4-fluorobenzylamine (6, 36.2 g,0.29 mol) were added, and the
mixture was heated un-der reflux for 10 h. The mixture was cooled
to 5 ◦C,water (750 mL) was added, and stirring was continuedfor
another hour. The product was collected by filtration,washed with
cold water (2 × 50 mL) and dried. Com-pound 7 (70.19 g, 98.8%) was
obtained as a pale-yellowsolid; m. p. 180 – 181 ◦C (lit.: 171 – 174
◦C [8]). – Rf = 0.31(hexane-THF, 3 : 1). – IR (KBr): ν = 3413s,
3368s, 3134m,1636s, 1608s, 1509m, 1491m, 1405m, 1375m, 1281s,
1255s,1172s, 1116m, 1014m, 774w cm−1. – UV/Vis (MeOH):λmax(logε) =
220 (3.72), 272 (3.49), 307 (3.25), 396 nm(3.97). – 1H NMR (400
MHz, [D6]DMSO): δ = 7.97 (d,J = 9.3 Hz, 1 H, 4-H), 7.42−7.36 (m, 2
H, 2 × 9-H),7.18−7.12 (m, 2 H, 2 × 10-H), 6.00 (d, J = 9.3 Hz, 1
H,5-H), 4.56 (d, J = 5.4 Hz, 2 H, 7-CH2) ppm. – 13C NMR(125 MHz,
CDCl3): δ = 161.3 (d, 1JC,F = 242.6 Hz, C-11),160.4 (C-6), 155.7
(C-2), 135.2 (d, 4JC,F = 3.0 Hz, C-8),134.5 (C-4), 129.7 (d, 3JC,F
= 7.8 Hz, C-9), 117.6 (C-3),115.1 (d, 2JC,F = 21.2 Hz, C-10), 102.3
(C-5), 43.1 (C-7)ppm. – 19F NMR (376 MHz, [D6]DMSO): δ = −115.9(tt,
3JF,H = 8.9 Hz, 4JF,H = 5.5 Hz) ppm. – MS ((+)-ESI):m/z (%) = 262.1
(100) [M+H]+, 285.1 (10) [M+Na]+. –C12H11FNO4 (262.24): calcd. C
54.96, H 4.23, N, 21.36;found C 54.72, H 4.29, N 21.31.
2-Amino-6-(4-fluorobenzylamino)-3-bromo-5-nitropyridine (8)
To a solution of 7 (20.00 g, 76.2 mmol) and ammoniumacetate
(0.58 g, 7.6 mmol) in a mixture of THF (150 mL)and acetonitrile
(150 mL) NBS (13.70 g, 77.0 mmol) wasadded at 0 ◦C in several small
portions, and stirring wascontinued at this temperature for 3 h.
The solvent was re-moved under reduced pressure, and the residue
subjectedto chromatography (silica gel 60,
chloroform-hexane-ethylacetate, 9 : 5 : 1) to yield 8 (15.66 g,
98.6%) as a yel-low solid; m. p. 143 – 144 ◦C. – Rf = 0.63
(chloroform-hexane-ethyl acetate, 9 : 5 : 1). – IR (KBr): ν =
3490m,3322m, 3091w, 2948w, 1598s, 1553m, 1508m, 1474m,1404m, 1273s,
1228s, 1124m, 1054w, 762m cm−1. – UV/Vis(MeOH): λmax(logε) = 226
(3.71), 281 (3.43), 403 nm(3.81). – 1H NMR (400 MHz, CDCl3): δ =
8.40 (s, 1 H,4-H), 7.32−7.27 (m, 2 H, 2× 9-H), 7.07−7.02 (m, 2 H,2×
10-H), 4.66 (d, J = 5.7 Hz, 2 H, 7-CH2) ppm. – 13CNMR (100 MHz,
CDCl3): δ = 162.5 (d, 1JC,F = 246.4 Hz,C-11), 156.2 (C-6), 153.8
(C-2), 137.5 (C-4), 133.5 (d,4JC,F = 3.4 Hz, C-8), 129.5 (d, 3JC,F
= 7.9 Hz, C-9), 120.3(C-3), 115.9 (d, 2JC,F = 21.5 Hz, C-10), 93.7
(C-5), 45.3 (C-7) ppm. – 19F NMR (376 MHz, [D6]DMSO): δ =
−115.9(tt, 3JF,H = 8.6 Hz, 4JF,H = 5.4 Hz) ppm. – MS ((+)-ESI):m/z
(%) = 341.1 (100) [M+H]+, 343.1 (92) [M+H]+. –C12H10BrFN4O2
(341.14): calcd. C 42.25, H 2.95, N, 16.42;found C 42.17, H 3.06, N
16.32.
Ethyl {2-amino-5-bromo-6-[(4-fluorobenzyl)amino]-pyridine-3-yl}
carbamate (10)
To a solution of 8 (10.00 g, 29.4 mmol) in EtOAc(150 mL) a
solution of ammonium acetate (15.72 g,294.0 mmol) in water (80 mL)
was added. Zinc powder(11.54 g, 176.4 mmol) was added in small
portions (keep-ing the temperature of the reaction below 35 ◦C).
After stir-ring for an additional hour, the mixture was filtered
througha small pad of Celite, washed with water (3× 50 mL) andbrine
(2× 50 mL) and dried (MgSO4). The mixture was fil-tered, and dry
triethylamine (4.16 g, 41.2 mmol) was added;the mixture was cooled
to 0 ◦C, and ethyl chloroformate(3.82 g, 35.2 mmol) was slowly
added. The mixture wasstirred for 2 h, water (70 mL) was added, the
phases wereseparated, and the organic phase was washed with wa-ter
(3× 50 mL) and brine (60 mL). The solvents were re-moved under
diminished pressure, and the residue sub-jected to chromatography
(silica gel 60, chloroform-ethylacetate, 9 : 1) to afford 10 (9.04
g, 80.5%) as a color-less solid; m. p. 146 – 147 ◦C. – Rf = 0.30
(chloroform-ethylacetate, 9 : 1). – IR (KBr): ν = 3431m, 3356m,
3293m,2885w, 1679s, 1638m, 1608m, 1530s, 1508s, 1438m, 1267s,1221m,
1153w, 1072m, 1011w, 816w, 500w cm−1. – UV/Vis(MeOH): λmax(logε) =
208 (3.94), 252 (3.59), 328 nm
-
1302 R. Csuk et al. · An Unprecedented Flupirtine Dimer
(3.48). – 1H NMR (400 MHz, [D6]DMSO): δ = 7.41−7.33(m, 3 H, 4-H,
2× 9-H), 7.12−7.06 (m, 2 H, 2× 10-H), 4.48(d, J = 6.1 Hz, 2 H,
7-CH2), 4.05 (q, J = 7.1 Hz, 2 H, 13-CH2), 1.21 (t, J = 6.9 Hz, 3
H, 14-CH3) ppm. – 13C NMR(100 MHz, [D6]DMSO): δ = 161.0 (d, 1JC,F =
241.6 Hz, C-11), 155.0 (C-12, C =O), 152.1 (C-2), 150.7 (C-6),
137.2 (d,4JC,F = 2.9 Hz, C-8), 137.2 (C-4), 129.3 (d, 3JC,F = 8.0
Hz,C-9), 114.7 (d, 2JC,F = 21.1 Hz, C-10), 107.9 (C-3), 87.2(C-5),
60.2 (C-13, CH2), 43.4 (C-7), 14.6 (C-14, CH3)ppm. – 19F NMR (376
MHz, [D6]DMSO): δ = −116.8 (tt,3JF,H = 8.9 Hz, 4JF,H = 5.7 Hz) ppm.
– MS ((–)-ESI): m/z(%) = 380.9 (83) [M−H]−, 383.0 (100) [M−H]−,
335.2 (19)[M−EtOH]−, 337.2 (19) [M−EtOH]−. – C15H16BrFN4O2(383.22):
calcd. C 47.02, H 4.21, N, 14.62; found C 46.85, H4.41, N
14.60.
Diethyl
6-bromo-5-[(4-fluorobenzyl)amino]-2-oxo-1H-imidazo[4,5-b]pyridine-1,3(2H)-dicarboxylate
(11)
Compound 11 (1.42 g, 10%) was obtained as a colorlesssolid; m.
p. 162 ◦C. – Rf = 0.63 (chloroform-ethyl acetate,9 : 1). – IR
(KBr): ν = 3409s, 2984w, 2938w, 1802s, 1726s,1622s, 1510s, 1427s,
1372s, 1334s, 1221s, 1148s, 1072m,1038m, 898w, 856m, 767m, 745m,
692m, 613w, 546w, 476wcm−1. – UV/Vis (MeOH): λmax(logε) = 258
(3.53), 337 nm(3.36). – 1H NMR (400 MHz, CDCl3): δ = 8.14 (s, 1 H,
4-H), 7.38−7.33 (m, 2 H, 2× 9-H), 7.02−7.96 (m, 2 H, 2× 10-H), 4.63
(s, 2 H, 7-CH2), 4.52−4.46 (m, 4 H, 14-CH2, 7-CH2), 1.45 (t, J =
7.1 Hz, 3 H, 18-CH3), 1.40 (t, J = 7.1 Hz,3 H, 15-CH3) ppm. – 13C
NMR (100 MHz, CDCl3): δ =162.3 (d, 1JC,F = 245.4 Hz, C-11), 151.3
(C-6), 149.9 (C-16,C=O), 148.2 (C-13, C=O), 146.0 (C-12, C=O),
138.1 (C-2), 135.0 (d, 4JC,F = 3.1 Hz, C-8), 129.7 (d, 3JC,F = 8.0
Hz,C-9), 126.8 (C-4), 115.5 (d, 2JC,F = 21.4 Hz, C-10), 111.5(C-3),
99.1 (C-5), 64.4 (C-17, CH2), 64.3 (C-14, CH2), 45.4(C-7), 14.3
(C-18, CH3), 14.2 (C-15, CH3) ppm. – 19FNMR (376 MHz, CDCl3): δ =
−120.1 (tt, 3JF,H = 8.9 Hz,4JF,H = 5.7 Hz) ppm. – MS ((+)-ESI): m/z
(%) = 480.9 (8)[M+H]+, 503.1 (100) [M+Na]+, 984.8 (48) [2M+Na]+.
–C19H18BrFN4O5 (481.04): calcd. C 47.42, H 3.77, N, 11.64;found C
47.31, H 3.93, N 11.49.
Diethyl
{6,6′-diamino-2,2′-bis[(4-fluorobenzyl)amino]-3,3′-bipyridine-5,5′-diyl}
biscarbamate (4)
Under argon a solution of 10 (10.00 g, 26.1 mmol)
andtriethylamine (7.92 g, 78.3 mmol) in dry THF (100 mL)at 60 ◦C
was added to a suspension of NiCl2 (0.34 g,2.6 mmol),
triphenylphosphane (5.46 g, 20.8 mmol), pow-dered Zn (5.12 g, 78.3
mmol), and triethylamine (7.92 g,78.3 mmol) in dry THF (100 mL).
The mixture was stirredfor 2 days at 60 ◦C, then the mixture was
poured onto thetop of a glass column containing silica gel. The
productwas eluted using a mixture of THF-diethyl ether (1 : 1).
After dilution with the double volume of hexane, the
pre-cipitated product was collected by filtration and washedwith a
mixture of hexane-THF-diethyl ether (2 : 1 : 1) andfinally dried
overnight. Re-precipitation from THF withhexane gave 4 (5.46 g,
69.0%) as a slightly off-whitesolid. – HPLC/DAD (LaChrom D-7000,
Merck-Hitachi):Nucleosil 100 – 5 C18−AB (Macherey-Nagel), 35 ◦C,
30%CH3CN/phosphate buffer (50 mM, pH = 2.8), 1 mL min−1,retention
time 12.5 min (retention time 1: 4.5 min); m. p.244 ◦C. – Rf = 0.58
(hexane-THF, 1 : 1). – IR (KBr):ν = 3392m, 3324s, 2983w, 2850w,
1682s, 1638m, 1586m,1528s, 1430s, 1384m, 1221s, 1157w, 1060m, 824w,
770w,602w, 577w, 501w cm−1. – UV/Vis (MeOH): λmax (logε)= 206
(3.83), 251 (3.41), 328 nm (3.37). – 1H NMR(500 MHz, [D6]DMSO): δ =
7.34−7.30 (m, 4 H, 4× 9-H),7.04−6.99 (m, 4 H, 4× 10-H), 6.99−6.95
(brs, 2 H, 2× 4-H), 4.47−4.36 (m, 4 H, 2× 7-CH2), 4.05 (brq, J =
7.0 Hz,4 H, 2× 13-CH2), 1.21 (brt, J = 6.2 Hz, 6 H, 2× 14-CH3)ppm.
– 13C NMR (125 MHz, [D6]DMSO): δ = 161.0 (d,1JC,F = 241.5 Hz,
C-11), 155.0 (C-12, C =O), 152.1 (C-6),151.9 (C-2), 137.5 (d, 4JC,F
= 2.8 Hz, C-8), 136.6 (C-4),129.4 (d, 3JC,F = 7.9 Hz, C-9), 114.6
(d, 2JC,F = 21.0 Hz, C-10), 107.5 (C-3), 104.8 (C-5), 60.1 (C-13,
CH2), 43.7 (C-7),14.6 (C-14, CH3) ppm. – 19F NMR (376 MHz,
[D6]DMSO):δ = −117.0 (tt, 3JF,H = 9.1 Hz, 4JF,H = 5.7 Hz) ppm. –
MS((+)-ESI): m/z (%) = 304.1 (22) [M+2H]2+, 607.2 (100)[M+H]+). –
C30H32F2N8O4 (606.52): calcd. C 59.40, H5.32, N, 18.47; found C
59.32, H 5.47, N 18.36.
Table 1. Crystallographic data for compound 11.
Identification code i2t0902Empirical formula
C19H18BrFN4O5Formula weight 481.28Crystal size, mm3 0.23 × 0.10×
0.09Temperature T , K 200(2)Wavelength λ , Å 0.71073Crystal
system; space group monoclinic; P21/nUnit cell dimensionsa, Å
4.901(1)b, Å 23.996(1)c, Å 16.943(1)β , deg 90.69(1)Volume V ,
Å3 1992.2(3)Z; calculated density Dcalcd., g cm−3 2;
1.61Absorption coefficient µ(MoKα ), mm−1 2.12θ range for data
collection, deg 6.22−55.99Limiting indices hkl ±6, −26→ 31, −19→
22Reflections collected / unique / Rint 9380 / 4644 / 0.0549Data/
refined parameters 4644 / 271Final indices R1 / wR2 [I > 2σ(I)]
0.0343 / 0.0740Funal indices R1 / wR2 (all data) 0.0749 /
0.0991Goodness of fit on F2 0.804Largest diff. peak / hole ∆ρfin
0.41 / −0.43(max / min), e Å−3
-
R. Csuk et al. · An Unprecedented Flupirtine Dimer 1303
Table 2. Selected bond lengths (Å) and bond angles (deg)
forcompound 11.
C(1)–N(1) 1.355(4) C(8)–C(13) 1.382(5)C(1)–N(4) 1.344(4)
C(9)–C(10) 1.387(6)C(1)–C(2) 1.423(4) C(10)–C(11) 1.342(6)C(2)–C(3)
1.381(5) C(11)–C(12) 1.341(6)C(2)–Br 1.892(3) C(11)–F
1.376(5)C(3)–C(4) 1.375(4) C(12)–C(13) 1.380(6)C(4)–C(5) 1.375(4)
C(14)–O(2) 1.202(3)C(4)–N(2) 1.410(4) C(14)–O(3) 1.312(4)C(5)–N(1)
1.316(4) C(14)–N(2) 1.398(4)C(5)–N(3) 1.422(4) C(15)–O(3)
1.466(4)C(6)–O(1) 1.196(4) C(15)–C(16) 1.508(5)C(6)–N(3) 1.409(4)
C(18)–O(5) 1.466(4)C(6)–N(2) 1.416(4) C(18)–C(19) 1.478(6)C(7)–N(4)
1.454(4) N(3)–C(17) 1.414(3)C(7)–C(8) 1.510(5) C(17)–O(4)
1.200(3)C(8)–C(9) 1.358(5) C(17)–O(5) 1.306(4)N(4)–C(1)–N(1)
118.1(3) O(2)–C(14)–O(3) 126.2(3)N(4)–C(1)–C(2) 121.7(3)
O(2)–C(14)–N(2) 120.7(3)C(1)–C(2)–Br 118.9(3) O(3)–C(14)–N(2)
113.1(2)C(3)–C(2)–Br 119.5(2) C(5)–N(1)–C(1) 116.7(3)C(3)–C(4)–N(2)
132.4(3) C(14)–N(2)–C(4) 122.9(2)C(5)–C(4)–N(2) 108.2(2)
C(14)–N(2)–C(6) 126.7(3)N(1)–C(5)–N(3) 126.8(3) C(5)–N(3)–C(17)
128.8(3)O(1)–C(6)–N(2) 127.7(3) C(6)–N(3)–C(17)
121.1(2)O(1)–C(6)–N(3) 127.5(3) C(1)–N(4)–C(7)
123.0(3)N(2)–C(6)–N(3) 104.8(2) O(4)–C(17)–O(5)
126.5(3)N(4)–C(7)–C(8) 112.6(3) O(4)–C(17)–N(3)
122.5(3)C(7)–C(8)–C(9) 121.6(3) O(5)–C(17)–N(3)
110.9(2)C(7)–C(8)–C(13) 120.5(3) C(14)–O(3)–C(15)
113.7(2)C(10)–C(11)–F 118.8(4) C(17)–O(5)–C(18)
116.5(2)C(12)–C(11)–F 118.4(4)
Table 3. Hydrogen bonds for compound 11 (Å and deg)a.
D–H···A d(D–H) d(H···A) d(D···A)
-
1304 R. Csuk et al. · An Unprecedented Flupirtine Dimer
[18] G. P. Zecchini, I. Torrini, M. P. Paradisi, J.
HeterocyclicChem. 1985, 22, 1061 – 1064.
[19] G. M. Sheldrick, SHELXS/L-97, Programs for Crys-tal
Structure Determination, University of Göttingen,Göttingen
(Germany) 1997. See also: G. M. Sheldrick,Acta Crystallogr. 2008,
A64, 112 – 122.
[20] J. Hassan, M. Sevignon, C. Gozzi, E. Schulz, M. Le-maire,
Chem. Rev. 2002, 102, 1359 – 1469.
[21] M. Tiecco, L. Testaferri, M. Tingoli, D. Chianelli,M.
Montanucci, Synthesis 1984, 736 – 738.
[22] A. S. Kende, L. S. Liebeskind, D. M. Braitsch, Tetrahe-dron
Lett. 1975, 3375 – 3378.
[23] M. Iyoda, H. Otsuka, K. Sato, N. Nisato, M. Oda, Bull.Chem.
Soc. Jpn. 1990, 63, 80 – 87.
[24] I. Colon, D. R. Kelsey, J. Org. Chem. 1986, 51, 2627
–2637.
[25] X. C. Tao, W. Zhou, Y. P. Zhang, C. Y. Dai, D. Shen,M.
Huang, Chinese J. Chem. 2006, 24, 939 – 942.
[26] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J.McMahon, D.
Vistica, J. T. Warren, H. Bokesch,S. Kenney, M. R. Boyd, J. Natl.
Cancer Inst. 1990, 82,1107 – 1112.
[27] K. Brandenburg, DIAMOND (version 3.0), Crys-tal and
Molecular Structure Visualization, CrystalImpact – K. Brandenburg
& H. Putz GbR, Bonn (Ger-many) 2004. See also:
http://www.crystalimpact.com/diamond/.
http://dx.doi.org/10.1002/jhet.5570220427http://dx.doi.org/10.1002/jhet.5570220427http://dx.doi.org/10.1107/S0108767307043930http://dx.doi.org/10.1107/S0108767307043930http://dx.doi.org/10.1107/S0108767307043930http://dx.doi.org/10.1107/S0108767307043930http://dx.doi.org/10.1021/cr000664rhttp://dx.doi.org/10.1021/cr000664rhttp://dx.doi.org/10.1246/bcsj.63.80http://dx.doi.org/10.1246/bcsj.63.80http://dx.doi.org/10.1021/jo00364a002http://dx.doi.org/10.1021/jo00364a002http://dx.doi.org/10.1002/cjoc.200690178http://dx.doi.org/10.1002/cjoc.200690178http://dx.doi.org/10.1093/jnci/82.13.1107http://dx.doi.org/10.1093/jnci/82.13.1107http://dx.doi.org/10.1093/jnci/82.13.1107http://dx.doi.org/10.1093/jnci/82.13.1107http://www.crystalimpact.com/diamond/http://www.crystalimpact.com/diamond/
Isolation, Structure, Synthesis and Cytotoxicity of an
Unprecedented Flupirtine Dimer1 Introduction2 Results and
Discussion3 Experimental Section3.1 Cell lines and culture
conditions3.2 Cytotoxicity assay 263.3 Synthesis and analysis3.4
2-Amino-6-(4-fluorobenzylamino)-3-nitropyridine (7)3.5
2-Amino-6-(4-fluorobenzylamino)-3-bromo-5-nitropyridine (8)3.6
Ethyl {2-amino-5-bromo-6-[(4-fluorobenzyl)amino]-pyridine-3-yl}
carbamate (10)3.7 Diethyl
6-bromo-5-[(4-fluorobenzyl)amino]-2-oxo-1H-imidazo[4,5-b]pyridine-1,3(2H)-dicarboxylate
(11)3.8 Diethyl
{6,6-diamino-2,2-bis[(4-fluorobenzyl)amino]-3,3-bipyridine-5,5-diyl}
biscarbamate (4)3.9 Crystal structure determination3.10
Acknowledgement